Nontypeable Haemophilus influenzae (NTHi) is an important etiological agent of otitis media (OM), one of the most common infectious illnesses of childhood in the United States. The molecular mechanism by which NTHi causes OM is not fully understood. Microbial infection-mediated deleterious inflammation is a hallmark of OM. It has been reported that interleukin 8 (IL-8), one of the important inflammatory mediators, is induced by NTHi and plays a significant role in OM pathogenesis. However, the bacterial factors of NTHi responsible for the inflammatory response and the cellular mechanisms involved remain largely elusive. Our preliminary studies indicate that the soluble cytoplasmic fraction of NTFE (NTHi SCF) strongly induces IL-8 secretion. In addition, multiple intracellular signaling pathways are activated in response to NTHi SCF and may involve NTHi-induced IL-8 up-regulation. The long-term goal of this study is to identify NTHi molecules responsible for deleterious inflammation in OM and to understand the cellular and molecular mechanisms by which NTHi soluble cytoplasmic molecules induce the production of inflammatory mediators. Based on our recent findings, we hypothesize that the soluble cytoplasmic molecules of NTHi induce IL-8 up-regulation through p38 and ERK MAPK pathways. To test this hypothesis, we will: (1) Characterize and purify NTHi molecules responsible for the intense IL-8 upregulation; (2) Determine whether NTHi induces IL-8 up-regulation through activation of the p38 MAPK pathway; (3) Determine the contribution of the ERK MAPK pathway in NTHi-induced IL-8 up-regulation. Significance: The establishment of an IL-8 expression assay in the laboratory will enable us to test the hypothesis quantitatively at molecular level. Accomplishment of these goals will provide critical and essential information about the molecular and cellular basis for pathogenesis of OM and rational targets for anti -inflammatory intervention.